The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Michael J Blackman - One of the best experts on this subject based on the ideXlab platform.
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the malarial serine protease sub1 plays an essential role in parasite liver stage development
PLOS Pathogens, 2013Co-Authors: Catherine Suarez, Katrin Volkmann, Ana Rita Gomes, Oliver Billker, Michael J BlackmanAbstract:Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter Merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage Merozoite biology are similar to those in the much better-studied blood stage Merozoites, little is known of the molecular players involved in liver stage Merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage Merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic Merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.
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malaria parasite cgmp dependent protein kinase regulates blood stage Merozoite secretory organelle discharge and egress
PLOS Pathogens, 2013Co-Authors: Christine R Collins, David Baker, Fiona Hackett, Malcolm Strath, Maria Penzo, Chrislaine Withersmartinez, Michael J BlackmanAbstract:The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular Merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing Merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct Merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.
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malaria parasite cgmp dependent protein kinase regulates blood stage Merozoite secretory organelle discharge and egress
PLOS Pathogens, 2013Co-Authors: Christine R Collins, David Baker, Fiona Hackett, Malcolm Strath, Maria Penzo, Chrislaine Withersmartinez, Michael J BlackmanAbstract:The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular Merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing Merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct Merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.
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global identification of multiple substrates for plasmodium falciparum sub1 an essential malarial processing protease
Infection and Immunity, 2011Co-Authors: Natalie Silmon C De Monerri, Fiona Hackett, Chrislaine Withersmartinez, Konstantinos Koussis, Helen R Flynn, Marta G Campos, Mark J Skehel, Michael J BlackmanAbstract:The protozoan pathogen responsible for the most severe form of human malaria, Plasmodium falciparum, replicates asexually in erythrocytes within a membrane-bound parasitophorous vacuole (PV). Following each round of intracellular growth, the PV membrane (PVM) and host cell membrane rupture to release infectious Merozoites in a protease-dependent process called egress. Previous work has shown that, just prior to egress, an essential, subtilisin-like parasite protease called PfSUB1 is discharged into the PV lumen, where it directly cleaves a number of important Merozoite surface and PV proteins. These include the essential Merozoite surface protein complex MSP1/6/7 and members of a family of papain-like putative proteases called SERA (serine-rich antigen) that are implicated in egress. To determine whether PfSUB1 has additional, previously unrecognized substrates, we have performed a bioinformatic and proteomic analysis of the entire late asexual blood stage proteome of the parasite. Our results demonstrate that PfSUB1 is responsible for the proteolytic processing of a range of Merozoite, PV, and PVM proteins, including the rhoptry protein RAP1 (rhoptry-associated protein 1) and the Merozoite surface protein MSRP2 (MSP7-related protein-2). Our findings imply multiple roles for PfSUB1 in the parasite life cycle, further supporting the case for considering the protease as a potential new antimalarial drug target.
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a multifunctional serine protease primes the malaria parasite for red blood cell invasion
The EMBO Journal, 2009Co-Authors: Konstantinos Koussis, Fiona Hackett, Chrislaine Withersmartinez, Hermann Bujard, Sharon Yeoh, Matthew A Child, Ellen Knuepfer, Luiz Juliano, Ute Woehlbier, Michael J BlackmanAbstract:The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter Merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of Merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in ‘priming' the Merozoite prior to invasion. The major protein complex on the Merozoite surface comprises three proteins called Merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the Merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria Merozoite and remodelling of its surface in preparation for erythrocyte invasion.
Michelle J Boyle - One of the best experts on this subject based on the ideXlab platform.
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sequential processing of Merozoite surface proteins during and after erythrocyte invasion by plasmodium falciparum
Infection and Immunity, 2014Co-Authors: Michelle J Boyle, Anthony N Hodder, Robin F Anders, Christine Langer, Joanne Chan, Ross L Coppel, James G BeesonAbstract:Plasmodium falciparum causes malaria disease during the asexual blood stages of infection when Merozoites invade erythrocytes and replicate. Merozoite surface proteins (MSPs) are proposed to play a role in the initial binding of Merozoites to erythrocytes, but precise roles remain undefined. Based on electron microscopy studies of invading Plasmodium Merozoites, it is proposed that the majority of MSPs are cleaved and shed from the surface during invasion, perhaps to release receptor-ligand interactions. In this study, we demonstrate that there is not universal cleavage of MSPs during invasion. Instead, there is sequential and coordinated cleavage and shedding of proteins, indicating a diversity of roles for surface proteins during and after invasion. While MSP1 and peripheral surface proteins such as MSP3, MSP7, serine repeat antigen 4 (SERA4), and SERA5 are cleaved and shed at the tight junction between the invading Merozoite and erythrocyte, the glycosylphosphatidylinositol (GPI)-anchored proteins MSP2 and MSP4 are carried into the erythrocyte without detectable processing. Following invasion, MSP2 rapidly degrades within 10 min, whereas MSP4 is maintained for hours. This suggests that while some proteins that are shed upon invasion may have roles in initial contact steps, others function during invasion and are then rapidly degraded, whereas others are internalized for roles during intraerythrocytic development. Interestingly, anti-MSP2 antibodies did not inhibit invasion and instead were carried into erythrocytes and maintained for approximately 20 h without inhibiting parasite development. These findings provide new insights into the mechanisms of invasion and knowledge to advance the development of new drugs and vaccines against malaria.
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new approaches to studying plasmodium falciparum Merozoite invasion and insights into invasion biology
International Journal for Parasitology, 2013Co-Authors: Michelle J Boyle, Danny W. Wilson, James G BeesonAbstract:Merozoite invasion of human red blood cells by Plasmodium falciparum is essential for blood stage asexual replication and the development of malaria disease. Despite this, many of the processes involved in invasion are poorly understood. Recent advances have been made in methods to isolate viable Merozoites for studies of invasion. The application of these approaches is providing new insights into the kinetics of invasion and Merozoite survival, as well as proteins and interactions involved in invasion, and will facilitate the development and testing of anti-Merozoite vaccines and the identification of invasion-inhibitory compounds with potential for drug development. This review discusses these recent advances and considers potential avenues for future research.
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isolation of viable plasmodium falciparum Merozoites to define erythrocyte invasion events and advance vaccine and drug development
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Danny W. Wilson, Michelle J Boyle, Jack S Richards, David T Riglar, Kevin K A Tetteh, David J ConwayAbstract:During blood-stage infection by Plasmodium falciparum, Merozoites invade RBCs. Currently there is limited knowledge of cellular and molecular invasion events, and no established assays are available to readily measure and quantify invasion-inhibitory antibodies or compounds for vaccine and drug studies. We report the isolation of viable Merozoites that retain their invasive capacity, at high purity and yield, purified by filtration of highly synchronous populations of schizonts. We show that the half-life of Merozoite invasive capacity after rupture is 5 min at 37 °C, and 15 min at room temperature. Studying the kinetics of invasion revealed that 80% of invasion events occur within 10 min of mixing Merozoites and RBCs. Invasion efficiency was maximum at low Merozoite-to-RBC ratios and occurred efficiently in the absence of serum and with high concentrations of dialyzed nonimmune serum. We developed and optimized an invasion assay by using purified Merozoites that enabled invasion-inhibitory activity of antibodies and compounds to be measured separately from other mechanisms of growth inhibition; the assay was more sensitive for detecting inhibitory activity than established growth-inhibition assays. Furthermore, with the use of purified Merozoites it was possible to capture and fix Merozoites at different stages of invasion for visualization by immunofluorescence microscopy and EM. We thereby demonstrate that processing of the major Merozoite antigen Merozoite surface protein-1 occurs at the time of RBC invasion. These findings have important implications for defining invasion events and molecular interactions, understanding immune interactions, and identifying and evaluating inhibitors to advance vaccine and drug development.
Robin F Anders - One of the best experts on this subject based on the ideXlab platform.
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sequential processing of Merozoite surface proteins during and after erythrocyte invasion by plasmodium falciparum
Infection and Immunity, 2014Co-Authors: Michelle J Boyle, Anthony N Hodder, Robin F Anders, Christine Langer, Joanne Chan, Ross L Coppel, James G BeesonAbstract:Plasmodium falciparum causes malaria disease during the asexual blood stages of infection when Merozoites invade erythrocytes and replicate. Merozoite surface proteins (MSPs) are proposed to play a role in the initial binding of Merozoites to erythrocytes, but precise roles remain undefined. Based on electron microscopy studies of invading Plasmodium Merozoites, it is proposed that the majority of MSPs are cleaved and shed from the surface during invasion, perhaps to release receptor-ligand interactions. In this study, we demonstrate that there is not universal cleavage of MSPs during invasion. Instead, there is sequential and coordinated cleavage and shedding of proteins, indicating a diversity of roles for surface proteins during and after invasion. While MSP1 and peripheral surface proteins such as MSP3, MSP7, serine repeat antigen 4 (SERA4), and SERA5 are cleaved and shed at the tight junction between the invading Merozoite and erythrocyte, the glycosylphosphatidylinositol (GPI)-anchored proteins MSP2 and MSP4 are carried into the erythrocyte without detectable processing. Following invasion, MSP2 rapidly degrades within 10 min, whereas MSP4 is maintained for hours. This suggests that while some proteins that are shed upon invasion may have roles in initial contact steps, others function during invasion and are then rapidly degraded, whereas others are internalized for roles during intraerythrocytic development. Interestingly, anti-MSP2 antibodies did not inhibit invasion and instead were carried into erythrocytes and maintained for approximately 20 h without inhibiting parasite development. These findings provide new insights into the mechanisms of invasion and knowledge to advance the development of new drugs and vaccines against malaria.
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plasmodium falciparum ring infected erythrocyte surface antigen is released from Merozoite dense granules after erythrocyte invasion
Infection and Immunity, 1991Co-Authors: Janetta G Culvenor, Karen P Day, Robin F AndersAbstract:Electron microscopy was used to study the fate of Plasmodium falciparum ring-infected erythrocyte surface antigen after Merozoite invasion by using postembedding immunolabeling. The antigen was localized to small dense granules located centrally or laterally in free Merozoites. In newly invaded erythrocytes, labeling was found in pockets of the parasitophorous vacuole space or in aggregates closely associated with the parasitophorous vacuole. These patterns indicate that ring-infected erythrocyte surface antigen is contained in Merozoite dense granules that are released after Merozoite invasion and not via apical rhoptry ducts at the time of Merozoite attachment. Images
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structural diversity in the plasmodium falciparum Merozoite surface antigen 2
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Jason Arthur Smythe, Karen P Day, Robin F Anders, Ross L Coppel, Rodger K Martin, A M J Oduola, David J KempAbstract:Abstract Antigens associated with the surface of Merozoites of the malaria parasite Plasmodium falciparum are directly accessible to immune attack and therefore are prime vaccine candidates. We have previously shown that one of the two known Merozoite surface antigens (Merozoite surface antigen 2; MSA-2) exhibits considerable sequence and antigenic diversity in different isolates. The sequences of MSA-2 from three isolates revealed a central domain composed of repeats that vary in number, length, and sequence, flanked in turn by nonrepetitive variable sequences and by conserved N- and C-terminal domains. We report here the sequences of a further four MSA-2 alleles, containing repetitive sequences that are related but not identical to each other. The seven alleles of MSA-2 can be divided into two distinct allele families on the basis of nonrepetitive sequences. Hybridization studies with repeat probes indicated that all of the 44 P. falciparum isolates examined contained repeat regions similar to those defined in known MSA-2 sequences.
Chrislaine Withersmartinez - One of the best experts on this subject based on the ideXlab platform.
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processing of plasmodium falciparum Merozoite surface protein msp1 activates a spectrin binding function enabling parasite egress from rbcs
Cell Host & Microbe, 2015Co-Authors: Sujaan Das, Christine R Collins, Chrislaine Withersmartinez, Abigail J Perrin, Nadine Hertrich, Matthew L Jones, Jean M Watermeyer, Elmar T Fobes, Stephen R Martin, Helen R SaibilAbstract:The malaria parasite Plasmodium falciparum replicates within erythrocytes, producing progeny Merozoites that are released from infected cells via a poorly understood process called egress. The most abundant Merozoite surface protein, MSP1, is synthesized as a large precursor that undergoes proteolytic maturation by the parasite protease SUB1 just prior to egress. The function of MSP1 and its processing are unknown. Here we show that SUB1-mediated processing of MSP1 is important for parasite viability. Processing modifies the secondary structure of MSP1 and activates its capacity to bind spectrin, a molecular scaffold protein that is the major component of the host erythrocyte cytoskeleton. Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and Merozoites lacking surface-bound MSP1 display a severe egress defect. Our results indicate that interactions between SUB1-processed Merozoite surface MSP1 and the spectrin network of the erythrocyte cytoskeleton facilitate host erythrocyte rupture to enable parasite egress.
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malaria parasite cgmp dependent protein kinase regulates blood stage Merozoite secretory organelle discharge and egress
PLOS Pathogens, 2013Co-Authors: Christine R Collins, David Baker, Fiona Hackett, Malcolm Strath, Maria Penzo, Chrislaine Withersmartinez, Michael J BlackmanAbstract:The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular Merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing Merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct Merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.
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malaria parasite cgmp dependent protein kinase regulates blood stage Merozoite secretory organelle discharge and egress
PLOS Pathogens, 2013Co-Authors: Christine R Collins, David Baker, Fiona Hackett, Malcolm Strath, Maria Penzo, Chrislaine Withersmartinez, Michael J BlackmanAbstract:The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular Merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing Merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct Merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.
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global identification of multiple substrates for plasmodium falciparum sub1 an essential malarial processing protease
Infection and Immunity, 2011Co-Authors: Natalie Silmon C De Monerri, Fiona Hackett, Chrislaine Withersmartinez, Konstantinos Koussis, Helen R Flynn, Marta G Campos, Mark J Skehel, Michael J BlackmanAbstract:The protozoan pathogen responsible for the most severe form of human malaria, Plasmodium falciparum, replicates asexually in erythrocytes within a membrane-bound parasitophorous vacuole (PV). Following each round of intracellular growth, the PV membrane (PVM) and host cell membrane rupture to release infectious Merozoites in a protease-dependent process called egress. Previous work has shown that, just prior to egress, an essential, subtilisin-like parasite protease called PfSUB1 is discharged into the PV lumen, where it directly cleaves a number of important Merozoite surface and PV proteins. These include the essential Merozoite surface protein complex MSP1/6/7 and members of a family of papain-like putative proteases called SERA (serine-rich antigen) that are implicated in egress. To determine whether PfSUB1 has additional, previously unrecognized substrates, we have performed a bioinformatic and proteomic analysis of the entire late asexual blood stage proteome of the parasite. Our results demonstrate that PfSUB1 is responsible for the proteolytic processing of a range of Merozoite, PV, and PVM proteins, including the rhoptry protein RAP1 (rhoptry-associated protein 1) and the Merozoite surface protein MSRP2 (MSP7-related protein-2). Our findings imply multiple roles for PfSUB1 in the parasite life cycle, further supporting the case for considering the protease as a potential new antimalarial drug target.
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a multifunctional serine protease primes the malaria parasite for red blood cell invasion
The EMBO Journal, 2009Co-Authors: Konstantinos Koussis, Fiona Hackett, Chrislaine Withersmartinez, Hermann Bujard, Sharon Yeoh, Matthew A Child, Ellen Knuepfer, Luiz Juliano, Ute Woehlbier, Michael J BlackmanAbstract:The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter Merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of Merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in ‘priming' the Merozoite prior to invasion. The major protein complex on the Merozoite surface comprises three proteins called Merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the Merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria Merozoite and remodelling of its surface in preparation for erythrocyte invasion.
James G Beeson - One of the best experts on this subject based on the ideXlab platform.
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sequential processing of Merozoite surface proteins during and after erythrocyte invasion by plasmodium falciparum
Infection and Immunity, 2014Co-Authors: Michelle J Boyle, Anthony N Hodder, Robin F Anders, Christine Langer, Joanne Chan, Ross L Coppel, James G BeesonAbstract:Plasmodium falciparum causes malaria disease during the asexual blood stages of infection when Merozoites invade erythrocytes and replicate. Merozoite surface proteins (MSPs) are proposed to play a role in the initial binding of Merozoites to erythrocytes, but precise roles remain undefined. Based on electron microscopy studies of invading Plasmodium Merozoites, it is proposed that the majority of MSPs are cleaved and shed from the surface during invasion, perhaps to release receptor-ligand interactions. In this study, we demonstrate that there is not universal cleavage of MSPs during invasion. Instead, there is sequential and coordinated cleavage and shedding of proteins, indicating a diversity of roles for surface proteins during and after invasion. While MSP1 and peripheral surface proteins such as MSP3, MSP7, serine repeat antigen 4 (SERA4), and SERA5 are cleaved and shed at the tight junction between the invading Merozoite and erythrocyte, the glycosylphosphatidylinositol (GPI)-anchored proteins MSP2 and MSP4 are carried into the erythrocyte without detectable processing. Following invasion, MSP2 rapidly degrades within 10 min, whereas MSP4 is maintained for hours. This suggests that while some proteins that are shed upon invasion may have roles in initial contact steps, others function during invasion and are then rapidly degraded, whereas others are internalized for roles during intraerythrocytic development. Interestingly, anti-MSP2 antibodies did not inhibit invasion and instead were carried into erythrocytes and maintained for approximately 20 h without inhibiting parasite development. These findings provide new insights into the mechanisms of invasion and knowledge to advance the development of new drugs and vaccines against malaria.
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new approaches to studying plasmodium falciparum Merozoite invasion and insights into invasion biology
International Journal for Parasitology, 2013Co-Authors: Michelle J Boyle, Danny W. Wilson, James G BeesonAbstract:Merozoite invasion of human red blood cells by Plasmodium falciparum is essential for blood stage asexual replication and the development of malaria disease. Despite this, many of the processes involved in invasion are poorly understood. Recent advances have been made in methods to isolate viable Merozoites for studies of invasion. The application of these approaches is providing new insights into the kinetics of invasion and Merozoite survival, as well as proteins and interactions involved in invasion, and will facilitate the development and testing of anti-Merozoite vaccines and the identification of invasion-inhibitory compounds with potential for drug development. This review discusses these recent advances and considers potential avenues for future research.